PROJECT SUMMARY Most bacterial viruses ("bacteriophages" or just "phage") are highly efficient in their ability to infect their often very specific bacterial hosts. In general, only one or a few viral particles are necessary to successfully infect one bacterium. As a consequence, there has been a long, unfulfilled hope that phages could be used as antibiotics. The need for alternative antibiotics is becoming ever more acute as common bacterial pathogens are developing resistant mutations, making it urgent to develop alternatives such as phage therapy. We have extensive experience in both structural and functional studies of phage T4. We now wish to expand this knowledge and make it available for medical applications. The plan is to analyze the structure and assembly of the capsid (Specific Aim 1), the DNA packaging machine (Specific Aim 2), the tail assembly (Specific Aim 3), assembly of the tail with head (Specific Aim 4) and the recognition of the host by the phage fibers (Specific Aim 5). Our primary tools will be molecular biology, protein chemistry, crystallography and electron microscopy. Molecular biology studies will produce pure samples in sufficient quantity for structural and functional studies. Crystallographic studies will be of protein components, providing three-dimensional information at near-atomic resolution. Cryoelectron microscopic reconstructions will provide three-dimensional data on the organization of the protein components within the virus. Combining these techniques will generate "pseudo-atomic" resolution structures of the virus at different stages of its life cycle. Our investigations will advance the use of bacteriophages for medical applications. For instance, among the problems encountered in clinical trials were the narrow host range of any one particular phage and the rapid degradation of phages when used as antibiotics in mammals. The host range is, in part, controlled by the viral enzymes that we will study, which are required to penetrate the host's cell wall and other defenses. Because of the repetitive nature of phage head capsids, they can be used to produce highly antigenic particles displaying epitopes of other viral pathogens thereby creating the potential for new vaccines. Other medical applications involve the powerful DNA packaging motor that might be used, for instance, in the delivery of genes in the development of gene therapy.